Miller, J. C. et al. Nat. Biotechnol. 37, 945–952 (2019).

Genome engineering with any type of exogenous nuclease needs to improve on two aspects if it is to realize its full potential for therapeutic applications: the editing efficiency must be high, to allow for biallelic or even multiplexed editing; and editing at unintended sites must be eliminated. Much progress has been made to increase the fidelity of currently used engineered nucleases such as Cas9, but most efforts to date have focused on tuning the affinity of the nuclease for its target DNA. Lam et al. instead add a point mutation to Fok1, the catalytic domain of zinc finger nucleases, and thereby slow the cleavage kinetics. This, in turn, reduces off-target activity. After developing an assay to detect very rare insertion/deletions below a signal of 0.01%, the researchers document a reduction in cutting at known off-target sites by up to 3,000-fold. They are able to target a T-cell receptor locus with 98% efficiency and no off-target cutting. This modified Fok1 domain could also be fused to other nucleases, such as Cas9, and enhance their specificity.